Herpes simplex virus gene expression is coordinately regulated and temporally controlled. The biosynthesis of early gene transcripts, eg the mRNA for thymidine kinase, is only catalyzed by host RNA polymerase after synthesis of functional immediate early gene products. When cloned immediate early gene are introduced into Hela cells along with a cloned tk gene, tk mRNA is faithfully transcribed. In the absence of immediate early gene sequences we detect little tk specific RNA. The experiments described in this grant application propose to use transient expression assays to define the immediate early genes that act in trans to activate transcription of tk gene. In other studies the sequences that code for the immediate early gene products will be modified in vitro and the effects of these mutations will be studied in a transient expression assay system. Our aim is to identify regulatory proteins, their functional domains and to determine if they interact with specific sequences in the virus chromosome.